Library of modified structural genes or capsid modified particles useful for the identification of viral clones with desired cell tropism

ABSTRACT

This invention relates to a library of nucleic acids comprising a multiplicity of expressible structural genes, preferably cap genes, from an eukaryotic virus, preferably of a parvovirus.

The present invention relates to libraries containing modifiedparvovirus cap genes useful for the identification of parvovirus capsidsable to transduce predefined cell types as well as to methods for theproduction thereof.

The control of the tropism of the vector (retargeting) represents acritical concern in the development of viral gene transfer systems forgene therapy: to allow efficient transfer of the therapeutic genes tothe target cells and to avoid transduction of undesired cell types.Efforts to achieve these goals led to the description of severalapproaches aimed to provide virions with the ability to interact withspecific cellular receptors. One of these approaches includes thecoupling of viral particles to receptor-binding molecules, which resultsin retargeted vectors with improved specificity. A big disadvantage ofthis technology is however that these retargeting molecules could detachfrom the capsids, restoring the natural tropism of the virions.

A different approach consists in the genetic modification of the viralcapsid or envelope proteins by site directed mutagenesis, mostly byinsertional mutagenesis.

For all these procedures, the critical step is the choice of functionalretargeting molecules. This problem has been adressed in several studiesby taking advantage of the phage display technology to screen largenumber of peptides for desired binding ability to specific receptors orcell types. However, the introduction of foreign molecules at the levelof the external structure of the vector is likely to disturb theintegrity of the viral particles, resulting in low titer or inefficientviral preparations. Moreover, these ligand peptides could completely orpartially lose their affinity for the aimed receptor once introduced inthe architecture of the vector. An elegant solution to this problem hasbeen proposed for adenoviral vectors (Pereboev A. et al. (2001) J Viro75(15), 7107-13). In this publication the expression of the Ad fiberknob on the surface of pJuFo phages is described, which allows thedisplay of polypeptide randomized sequences in a context that mimics themicroenvironment of the destination vector. However, a physiologicallimitation of phage-based libraries cannot be overcome: the moleculescan be selected exclusively on the basis of their binding ability, whileoptimization of the uptake and processing of the viral particles insidethe cell cannot be pursued.

Parvoviruses and especially the Adeno-associated virus (AAV) havereceived increasing attention as a vector for gene therapy because oftheir non-pathogenicity, their low immunogenicity, and their ability toinfect both dividing and non-dividing cells and to facilitate long termexpression of the therapeutic genes. Furthermore, AAV is able tointegrate site specifically into the genome of the infected cell withoutimpairing any cellular function.

A great disadvantage of the use of parvoviruses and other viruses ingene therapy is the fact that these viruses are only able to efficientlytransduce specific cell types, while other cell types are resistantagainst a parvovirus infection. Especially, AAV2 is only able totransduce cells having heparan sulfate proteoglycan (HSPG) on theirsurface (Girod A. et al.(1999) Nature Medicine 5-9, 1052-56). Asmentioned above, the control of the viral tropism is crucial for theeffective transfer of the therapeutic genes to the target cells and forthe prevention of a transduction of undesired cell types.

In the publication Girod A. et al.((1999) Nature Medicine 5-9, 1052-56)it was recently demonstrated that it is possible to modify the externalstructure of Adeno-associated virus in order to provide the capsid withthe ability to interact with cellular receptors that are not recognizedby the wild type virus. This procedure can be performed by insertion ofa ligand sequence at the level of a specific site of the viral capsidprotein, that is presented on the external surface of the capsid. As onesuitable locus, the amino acidic position 587 of the major viral capsidprotein (VP1) of AAV2 was identified. The insertion at this site of theL14 sequence, an RGD motif containing peptide (a portion of the lamininfragment P1), provided the so obtained mutant AAV (L14-AAV) with theability to infect B16F10 cells. The B 16F10 cells express an integrinand are, due to the lack of expression of heparan sulfate proteoglycansat the outer cellular membrane, resistant to infection with wtAAV2.

To date, no system exists which allows the fast identification ofmutated viruses, especially parvoviruses which are able to infect onecell type but are unable to infect other cell types. Such a system ishighly required for obtaining cell type specific parvoviruses to be usedin gene therapy.

Consequently, it is the object of the present invention to providesystems for the identification of virus, especially parvovirus mutantswhich are able to infect other cell types than the corresponding wildtype viruses. Furthermore, it is the object of the present invention toprovide methods for the preparation of such systems.

The present invention relates therefore in a first subject matter to amethod for the production of a library of nucleic acids comprising amultiplicity of expressible structural genes from at least oneeukaryotic virus, comprising the steps of:

-   -   a) providing a set of nucleic acids, each encoding at least one        structural gene from a eukaryotic virus and comprising a        suitable packaging sequence, and    -   b) inserting a first insert (1) into the structural gene.

According to the present invention, the expression “structural gene”relates to a gene encoding one or more proteins, preferably structuralproteins of a viral capsid either of a non-enveloped or an envelopedvirus or to gene encoding one or more proteins, preferably structuralproteins of a viral shell of enveloped viruses.

The invention further relates to a library of nucleic acids comprising amultiplicity of expressible structural genes from at least oneeukaryotic virus, obtainable by the above method.

The library of the invention contains a multiplicity of nucleic acidswith different structural genes which may be expressed in order to forminfectious viral particles with a tropism for different cell types.Given a certain cell type, it is therefore possible to screen thelibrary for mutant viruses, especially parvoviruses which are able toinfect that specific cell type.

According to the invention, the term “capsid protein” means a proteinencoded by a cap gene, whereas “functional capsid protein” means acapsid protein of a virus able to infect at least one host cell.

In case of AAV, the capsid protein may be VP1, VP2 or VP3, for otherparvoviruses names and numbers of the capsid proteins may differ.

According to the invention, the term “packaging sequence” means acis-acting nucleic acid sequence that mediates the packaging of anucleic acid into a viral capsid. For parvoviruses e.g. it is known inthe art, that the so called “inverted terminal repeats” (ITRs) that arelocated at the 5′ and 3′ end of the linear viral genome have thisfunction.

According to a preferred embodiment, the structural genes are from anenveloped virus such as a retrovirus, lentivirus, herpes virus, e.g.HSV1, HSV2, EBV, Varizella zoster virus, human herpes virus 1, 2, 3, 4,7 or 8.

According to another preferred embodiment, the structural genes are capgenes, preferably from a non-enveloped virus such as parvovirus oradenovirus. In this case, the cap genes encode for one or more capsidproteins

According to the present invention, the expression “cap gene” relates toa gene encoding one or more proteins of a viral capsid either of anon-enveloped or an enveloped virus.

More preferably, the cap genes are from a parvovirus selected from thegroup consisting of Adeno-associated Virus (AAV), Canine Parvovirus(CPV), MVM, B19, H1, AAAV (Avian AAV) or GPV (goose parvovirus).

Most preferred, the cap genes are from an AAV, e.g. AAV1, AAV2, AAV3,AAV4, AAV5 or AAV6.

In a preferred embodiment, the library obtainable by the method of theinvention has a multiplicity of viral mutants that is greater than 10²,preferably greater than 10⁵, especially greater than 10⁶ and, in anotherpreferred embodiment, a multiplicity of expressible structural genes,preferably cap genes that is greater than 10², preferably greater that10⁵, especially greater that 10⁶.

In a preferred embodiment, the set of nucleic acids is derived from onenucleic acid. In this case, the library is constituted of a multiplicityof nucleic acids which are, apart from the insert, almost identical.However, it is also included within the present invention that the setof nucleic acids may be derived from different nucleic acids encodingstructural genes. In this case, it is preferred that the nucleic acidsare derived from one virus.

According to a preferred embodiment, one insert (1) is inserted into thestructural gene. However, it is also included within the presentinvention that more than one inserts (1) are inserted. Preferably two,three or up to six inserts (1) are inserted into the structural gene.The insertion may, dependent on the insertion site, lead to an aminoacid insertion in one or more structural proteins, preferably capsidproteins, e.g. VP1, VP2 and/or VP3 in the case of AAV. In this contextit should be noted that, in the case of parvoviruses, parvovirus capsidproteins are encoded by only one cap gene by overlapping reading frames.

In a preferred embodiment of the present invention a sequence of thestructural gene is removed by inserting insert (1).

In a further preferred embodiment, the removed sequence comprises or ispart of an insert (2) inserted into the structural gene before step (a).

As a preferred embodiment the method further comprises an initial step,wherein the structural gene is modified to render the structural genenon-functional. This can be achieved i.e. by inserting insert (2). By areplacement of insert (2) with insert (1) in step a) of the method,potentially functional structural genes will be formed. In the case ofparvoviruses, this additional step leads to a reduced number ofparvovirus virions that have a capsid built from viral capsid proteinsnot encompassing insert (1) and therefore enables the formation oflibraries with high titers.

Consequently, in a more preferred embodiment, insert (2) prevents theformation of a functional structural protein, preferably a capsidprotein, preferably by containing a stop codon. Furthermore, insert (2)may shift the open reading frame or may introduce additional amino acidswhich disturb the formation of functional capsids or their infectionbiology at any further step.

In a preferred embodiment, insert (1) and/or insert (2) contains furtherat least two restriction sites, preferably one at its 3′-end and one atits 5′-end.

According to a preferred embodiment, insert (2) is at least partiallyreplaced by insert (1), whereby the prevention of the formation offunctional capsids is—at least for some capsids—abolished, in apreferred embodiment by removing the stop codon.

According to a preferred embodiment, the number of nucleotides of insert(1) and/or insert (2), preferably of insert (1) and insert (2), is threeor a multiple of three.

According to a preferred embodiment, insert (1) is inserted at a regionof the cap gene encoding amino acids on the surface of the capsidprotein.

In preferred embodiments, the virus is AAV2 and insert (1) is insertedafter a nucleic acid corresponding to a site within the first aminoterminal amino acids 1 to 50, or corresponding to amino acid positions261, 381, 447, 534, 573, and/or 587 of the capsid protein VP1,preferably corresponding to amino acid position 447 or 587.

In additional preferred embodiments, insert (1) is inserted in nucleicacids corresponding to the adjacent 5 amino acid of the above indicatedinsertion sites, as these amino acid stretches represent loops of theAAV2-capsid and therefore are located on the surface of the capsidprotein. It is possible for the person skilled in the art to identifycorresponding loops and insertion site for other parvoviruses by knowntechniques such as sequence alignment, three-dimensional structureanalysis, protein folding, hydrophobicity analysis (Girod A et al, 1999supra).

Especially preferred insertion sites are within the stretches consistingof amino acids (aa)

-   -   i) aa 261-270, especially aa 269,    -   ii) aa 324-331,    -   iii) aa 380-383, especially aa 381,    -   iv) aa 447-460, especially aa 447 and 451-460,    -   v) aa 484-503, preferably aa 484-499, especially aa 484, 487 or        aa 494-499,    -   vi) aa 507-514, especially aa 507, 509 or 514,    -   vii) aa 527-534, especially 527-529, 532 or 534,    -   viii) aa 548-556,    -   ix) aa 572-575, especially 573,    -   x) aa 581-595, especially 585, 587, 588 or 594.

The numbering of amino acids relates to the VP1 protein of AAV2 (Girod Aet al, 1999 supra).

Stretches v) and vi) are especially preferred.

The insert (1) inserted into the nucleic acid my be identical for allstructural genes where an insert (1) is inserted. However, it ispreferred that insert (1) is different or at least potentially differentfor all structural genes where an insert (1) is inserted.

In preferred embodiments, insert (1) is randomly or partially randomlygenerated. This means that the insert (1) introduced in one structuralgene is potentially different from the insert (1) inserted in anotherstructural gene, although it is theoretically possible that two inserts(1) are identical.

When the inserted nucleic acid sequences are randomly generated, in apreferred embodiment the codons NNN, NNB or NNK (N=A,C,G or T; B=C,G orT; K=G or T) are used. Furthermore, the inserted nucleic acid sequencesmay be partially randomly generated, especially using codons with one,two or three fixed nucleotides. The length of such insertions may bepreferably at least 3 nucleotides, preferably at least 9, especially atleast 18 nucleotides.

In a preferred embodiment, insert (1) may contain, in addition to therandomly or partially randomly generated sequences, a further stretch ofat least one codon upstream and/or downstream of the randomized orpartially randomized nucleic acid sequences, preferably of one or two orthree codons coding for Ala, Gly, Leu, Ile, Asp and/or Arg, especiallyan insertion of three codons for Ala upstream and two codons for Aladownstream of the randomized or partially randomized nucleic acidsequences.

In a preferred embodiment, insert (1) does not contain any stop codons.This can be achieved by not having an A or G at the third position ofthe codons of insert (1).

Furthermore, the library obtainable by the method of the invention mayhave the features as defined below for the library of nucleic acidscomprising a multiplicity of expressible structural genes from at leastone eukaryotic virus.

In a further subject matter, this invention relates to a library ofnucleic acids comprising a multiplicity of expressible structural genes,preferably cap genes, from an eukaryotic virus, preferably of aparvovirus, especially dependoviruses such as Adeno-associated virus ora canine parvovirus (CPV) as well as autonomous parvoviruses such as H1,MVM (minute virus of mice) or B19, AAAV or GPV.

Said library of nucleic acids may encode eucaryotic viruses with amultiplicity of modifications of the virion's external structure and/orof the corresponding (encoding) genetic information. The library has apreferred multiplicity of viral mutants that is greater than 10²,preferably greater that 10⁵, especially greater that 10⁶.

In a further embodiment, the library of nucleic acids has a multiplicityof expressible structural genes, preferably cap genes that is greaterthan 10², preferably greater that 10⁵ especially greater that 10⁶.

The library may be in the form of a linear nucleic acid, a plasmid, aviral particle or a viral vector, e.g. a recombinant AAV, Adenovirus orHerpes Simplex Virus vector.

In a preferred embodiment, the nucleic acid may additionally comprisepackaging sequences (e.g. AAV ITRs) and expressible genes providingnecessary functions for replication and packaging of virions (e.g.non-structural genes for parvoviruses such as AAV rep gene).

These sequences, genes or functions may be provided in cis (meaning onthe same construct as the packaging sequences and the capsid proteinsencoding genes) or in trans (meaning on a different construct) However,the packaging sequences must be provided in cis.

In a preferred embodiment, the nucleic acid is DNA.

In preferred embodiments, the cap gene as well as packaging sequencessuch as ITRs and genes providing necessary functions for replication andpackaging of virions, such as the rep gene, are derived fromparvoviruses, preferably from dependoviruses such as AAV or CPV,especially from one of the AAV serotypes from the group comprising AAV1,AAV2, AAV3, AAV4, AAV5 and AAV6 or from autonomous parvoviruses such asH1, MVM, B19, AAAV or GPV.

Another preferred embodiment relates to a library, wherein the AAV capgene is derived from the AAV cap gene encoded in plasmid pWT99oen (seeFIG. 1 and Example 1).

The multiplicity of nucleic acid sequences are inserted into at leastone site of the structural gene, preferably the cap gene, wherein thenumber of inserted nucleotides is three or a multiple of three.According to preferred embodiments, the multiplicity of nucleic acidsequences are inserted into one, two or three sites of the structuralgene, preferably the cap gene.

The inserted nucleic acid sequences are preferably randomly generated,especially using NNN codons, NNB codons or NNK codons (N=A,C,G or T;B=C,G or T; K=G or T). Furthermore, the inserted nucleic acid sequencesmay be partially randomly generated, especially using codons with one,two or three fixed nucleotides.

In a further preferred embodiment, a second or further insert (1) maycontain non randomized codons for amino acid stretches of choice. Thishas the advantage that one can simultaneously screen for expressiblestructural genes with a wanted property by inserting a randomized insert(1) and one or more further inserts (1) at different sites to change theproperties of such an expressible structural gene.

For example, if one has already identified an insert (1) that codes fora peptide and leads to- a retargetedvector-or-a-vector-with-other-wanted properties, one can use this insertat a specific site and use a randomized insert (1) at another site toscreen for a vector with other, enhanced properties. This procedure canbe repeated for several or all known potential insertion sites.

Furthermore, one can combine the insertion of a randomized insert (1)with the insertion of further fixed inserts (1) preferably at known orpresumed epitopes to change the immunogenicity of the vector. Within thescope of this invention. it was shown that either by using a randomizedinsert (1) and screening for a vector with an increased infectivity orspecificity for a specific cell type (rAAV-587/Mec, example 7) or byinserting a fixed insert (rAAV-587/L14, example 7) one can abolish orreduce the neutralizing effects of antibodies. Therefore, the inventioncan also be used to make or screen for vectors that have a reducedbinding to antibodies (monoclonal or polyclonal antibodies/sera) and/orto have the ability to escape neutralizing antibodies and therefore areable to escape from an immune response in a patient.

In a preferred embodiment, the length of such insertions is at least 3nucleotides, preferably at least 9, especially at least 18 nucleotides.

The inserted nucleic acid sequences may have been inserted usingstandard restriction endonucleases, recombination systems, e.g. thegateway or the cre/lox recombination system or polymerase chain reactiontechniques, e.g. using degenerated primers.

The inserted nucleic acid sequences shall lead to an insertion of aminoacids into at least one viral capsid protein, i.e. in the case of AAVinto VP1, VP2 and/or VP3 structural protein, preferably at a site thatis located on the surface of the capsid of the virion.

The inserted nucleic acid sequences may be inserted at any site withinthe first amino terminal amino acids 1 to 50 of VP1, after correspondingamino acid positions 261, 381, 447, 534, 573, and/or 587 of VP1,preferably after amino acid position 447 or 587. The numbering of theamino acids relates to the position within VP1. For the avoidance ofdoubt, corresponding sites of VP2 and VP3 of course have a differentnumber.

For AAV, this means that the inserted nucleic acid sequences may beinserted after a nucleic acid corresponding to any site within the firstamino terminal amino acids 1 to 50 of VP1, or corresponding to aminoacid positions 261, 381, 447, 534, 573, and/or 587 of VP1, preferably toamino acid position 447 or 587. The numbering of the amino acids relatesto the position within VP1. For the avoidance of doubt, correspondingsites of VP2 and VP3 of course have a different number.

In additional preferred embodiments, insert (1) is inserted in nucleicacids corresponding to the adjacent 5 amino acid of the above indicatedinsertion sites, as these amino acid stretches represent loops of theAAV2-capsid and therefore are located on the surface of the capsidprotein. It is possible for the person skilled in the art to identifycorresponding loops and insertion site for other parvoviruses by knowntechniques such as sequence alignment, three-dimensional structureanalysis, protein folding, hydrophobicity analysis (Girod A et al, 1999supra)

The cap genes may according to preferred embodiments in addition have atleast one further mutation being for example at least one pointmutation, at least one internal deletion, insertion and/or substitutionof one or several amino acids or at least one N- or C-terminal deletion,insertion and/or substitution of one or several amino acids, or acombination of these mutations, preferably a mutation inhibitingheparansulfate proteoglycan, integrins and/or Fibroblast Growth FactorReceptor (FGFR) binding. These additional specific mutations areespecially advantageous, since they reduce the infectivity of the virionfor a large number of its natural host cells.

Such further mutations can be used for an additional modification ofinfectivity of the Cap protein/virion, for a reduction of an infectionnot mediated by AAV e.g. by reducing or abolishing binding for cellularreceptors, or for a changed immunogenicity of the Cap protein/virion byreducing or abolishing the affinity to antibodies especially escapingfrom neutralizing antibodies.

Furthermore, such cap genes may have a further constant insertion of atleast one codon upstream and/or downstream of the insertion sites of therandomized nucleic acid sequences, preferably of one or two or threecodons coding for Ala, Gly, Leu, Ile, Asp and/or Arg, especially aninsertion of three Ala upstream and two Ala downstream of the insertionsite.

Furthermore this invention relates to a library of virions, especiallyparvovirus virions, with capsid protein modifications.

In a preferred embodiment of the invention, the library of virionscontains particles containing the genetic information necessary togenerate viral progeny.

A preferred embodiment is a library of said virions, where each particlecontains the genetic information necessary to generate viral progeny.

In a particularly preferred embodiment of the invention said library ofvirions is generated by using any of the above mentioned nucleic acids.

A further embodiment of this invention is a cap gene that comprises atleast one recombination site within the cap gene, e.g. for the Gatewayor cre/lox system, preferably after amino acid position 587 of VP1wherein the inserted nucleic acid sequences are inserted at any sitewithin the first amino-terminal amino acids 1 to 50 of VP1, aftercorresponding amino acid positions 261, 381, 447, 534, 573, and/or 587of VPI, preferably after amino acid position 447 or 587.

Especially preferred insertion sites are within the stretches consistingof amino acids (aa)

-   -   i) aa 261-270, especially aa 269,    -   ii) aa 324-331,    -   iii) aa 380-383, especially aa 381,    -   iv) aa 447-460, especially aa 447 and 451-460,    -   v) aa 484-503, preferably aa 484-499, especially aa 484, 487 or        aa 494-499,    -   vi) aa 507-514, especially aa 507, 509 or 514,    -   vii) aa 527-534, especially 527-529, 532 or 534,    -   viii) aa 548-556,    -   ix) aa 572-575, especially 573,    -   x) aa 581-595, especially 585, 587, 588 or 594.

The numbering of amino acids relates to the VP1 protein of AAV2 (Girod Aet al, 1999 supra).

Stretches v) and vi) are especially preferred.

This means that a further subject matter of this invention is a cap genethat comprises at least one recombination site within the cap gene,preferably for the Gateway or cre/lox system. For AAV, the recombinationsite may be inserted after a nucleic acid corresponding to any sitewithin the first amino terminal amino acids 1 to 50 of VP1, orcorresponding to amino acid positions 261, 381, 447, 534, 573, and/or587 of VP1, preferably to amino acid position 447 or 587. The numberingof the amino acids relates to the position within VP1. For the avoidanceof doubt, corresponding sites of VP2 and VP3 of course have a differentnumber. This cap gene of the invention can be used as starting materialfor the method of the invention for producing a parvovirus library.

Furthermore the cap gene may comprise at least one endonucleaserestriction site or polylinker that is not present in the respectivewildtype gene site useful for the insertion at any site within the firstamino-terminal amino acids 1 to 50 of VP1, after corresponding aminoacid positions 261, 381, 447, 534, 573, and/or 587 of VP1, preferablyafter amino acid position 447 or 587.

This means that the cap gene may comprise at least one endonucleaserestriction site or polylinker that is not present in the respectivewildtype gene. In the case of AAV2, the restriction site may be insertedafter a nucleic acid corresponding to any site within the firstamino-terminal amino acids 1 to 50 of VP1, or corresponding to aminoacid positions 261, 381, 447, 534, 573, and/or 587 of VP1, preferably toamino acid position 447 or 587.

In a preferred embodiment, the endonuclease restriction site orpolylinker may further contain a stop codon.

This will provide cap genes that do not contain an insertion with atranslation stop signal that will lead to defective capsid proteins andtherefore to no wild type virus production during the generation of thelibrary.

In a preferred embodiment, the cap gene of the invention may furtherhave at least one mutation, preferably at least one point mutation, atleast one internal deletion, insertion and/or substitution of one orseveral amino acids or at least one N- or C-terminal deletion, insertionand/or substitution of one or several amino acids, or a combination ofthese mutations.

Furthermore, such cap gene may have a further constant insertion of atleast one codon upstream and/or downstream of the insertion sites of therandomized nucleic acid sequences, preferably of one or two or threecodons coding for Ala, Gly, Leu, Ile, Asp and/or Arg, especially aninsertion of three Ala upstream and two Ala downstream of the insertionsite.

In a further preferred embodiment, the cap gene may contain nonrandomized codons for amino acid stretches of choice. This has theadvantage that one can simultaneously screen for expressible structuralgenes with a wanted property by inserting a randomized insert and one ormore further inserts at different sites to change the properties of suchan expressible structural gene.

For example, if one has already identified an insert that codes for apeptide and leads to a retargeted vector or a vector with other wantedproperties, one can use this insert at a specific site and use arandomized insert at another site to screen for a vector with other,enhanced properties. This procedure can be repeated for several or allknown potential insertion sites.

Furthermore, one can combine the insertion of a randomized insert withthe insertion of further fixed inserts preferably at known or presumedepitopes to change the immunogenicity of the vector. Within the scope ofthis invention it was shown that either by using a randomized insert andscreening for a vector with an increased infectivity or specificity fora specific cell type (rAAVö-587/Mec, example 7) or by inserting a fixedinsert (rAAV-587/L14, example 7) one can abolish or reduce theneutralizing effects of antibodies. Therefore, the invention can also beused to make or screen for vectors that have a reduced binding toantibodies (monoclonal or polyclonal antibodies/sera) and/or to have theability to escape neutralizing antibodies and therefore are able toescape from an immune response in a patient.

Therefore, in a most preferred embodiment, the cap gene of the inventioncontains an insert with

-   -   a) a restriction site or a recombination site;    -   b) one or more codons encoding further amino acids, preferably        Ala, Gly, Leu, Ile, Asp and/or Arg; and    -   c) a sequence stretch preventing formation of functional capsid        proteins, preferably a stop codon.

A further subject matter of this invention is the nucleic acid encodinga cap gene with a sequence of the plasmid pWT99oen (FIG. 1, sequencegiven and Example 1).

A further subject matter of this invention is a nucleic acid encoding acap gene, wherein such cap gene has an insertion leading to additionalamino acids comprising an RGD or DDD motif, preferably an RGDXP or DDDXPmotif, especially an RGD motif that is not present in human proteins,excluding the insertion AGTFALRGDNPQG. Such cap gene may have theinsertion corresponding to RGDXXXX, RGDXPXX, BDDXPXX, RGDAVGV orRGDTPTS, GKLFVDR, RDNAVVP, GENQARS, RSNGVVP, RSNAVVP or NSVRAPP.

The invention further relates to Cap proteins encoded by the above capgenes of the invention.

Another embodiment of this invention is a nucleic acid encoding a capgene, wherein such cap gene has an insertion of the nucleotidic sequenceGANGANNACNNNNCNANNANN (N=A,C,G or T) or an insertion comprising thatsequence.

The inserted nucleic acid sequences may be inserted at any sitecorresponding to the first amino-terminal amino acids 1 to 50 of VP1,after corresponding amino acid positions 261, 381, 447, 534, 573, and/or587 of VP1, preferably after amino acid position 447 or 587.

This means that in the case of AAV2, the nucleic acid of the inventionmay be inserted after a nucleic acid corresponding to any site withinthe first amino-terminal amino acids 1 to 50 of VP1, or corresponding toamino acid positions 261, 381, 447, 534, 573, and/or 587 of VP1,preferably to amino acid position 447 or 587.

In a preferred embodiment, the cap gene of the invention has at leastone mutation leading to preferably at least one point mutation, at leastone internal deletion, insertion and/or substitution of one or severalamino acids or at least one N- or C-terminal deletion, insertion and/orsubstitution of one or several amino acids, or a combination of thesemutations.

In a further preferred embodiment, such cap gene may have a furtherconstant insertion of at least one codon upstream and/or downstream ofthe insertion sites of the randomized nucleic acid sequences, preferablyof one or two or three codons coding for Ala, Gly, Leu, Ile, Asp and/orArg, especially an insertion of three Ala upstream and two Aladownstream of the insertion site.

The invention further relates to the use of a nucleic acid of theinvention encoding a cap gene for the preparation of a library ofnucleic acids comprising a multiplicity of expressible cap genes from atleast one eukaryotic virus, preferably a parvo-virus.

Further embodiments of this invention are vector constructs, bacteria orcells comprising any of the previously mentioned cap genes orconstructs.

A further embodiment of this invention is a method for the selection ofa recombinant virion with an increased infectivity or specificity for aspecific cell type comprising the steps of

-   -   i) providing at least one first cell with a vector construct        comprising at least one nucleic acid from the library of the        invention together with a second nucleic acid (especially        packaging sequences such as AAV ITRs and one or more genes        providing non-structural functions such as replication and        packaging, for example functions of an AAV Rep protein)        necessary for the packaging of a virion;    -   ii) providing such first cell with necessary cellular, viral,        physical and/or chemical helper functions for the packaging of        virions if necessary;    -   iii) incubating such first cell under suitable conditions for        the packaging of virions and collecting produced virions by such        first cell;    -   iv) infecting at least one second cell with such collected        virions;    -   v) providing such second cell with necessary cellular, viral,        physical and/or chemical helper functions for the packaging of a        virion;    -   vi) incubating such second cell under suitable conditions for        the packaging of virions and collecting produced virions by such        second cell;        whereas steps iv) to vi) can be repeated several times.

The non-structural functions as for example the Rep protein can beprovided in cis or in trans.

Furthermore, the first cell and the second cell can be of the same kindor type.

A further embodiment of the invention is a method for the selection of arecombinant virion with an increased infectivity or specificity for aspecific cell type, that at the same time has a reduced or noinfectivity for another cell type. To achieve such negative selectionthe above method additionally comprises the steps

-   -   vii) infecting at least one third cell (that shall not be        infected) with the collected virions, whereas such third cell is        not permissive fur such virions, and    -   viii) collecting the virions that did not infect such third        cell.

Using these additional steps virions that are able to infect such thirdcells enter the cells but do not replicate within these cells due to thenon-permissiveness of the cells. Therefore such virions are depletedfrom the library. Also these steps can be repeated on the same ordifferent cell types.

Non-permissive cells can be obtained for both helper dependent andhelper independent virions by not providing such third cell with allnecessary cellular, viral, physical and/or chemical functions for thepackaging of virions. One can also use drugs that inhibit viralreplication and/or packaging but not infection such as acilovir for HSV.Furthermore one can use virions that have been made replicationincompetent by mutations so that a cell has to provide a certainfunction to be permissive again.

Furthermore, this invention relates to a method for the identificationof a mutant cap gene leading to virions having an increased infectivityor specificity for a specific cell type comprising the previous stepsand in addition the, step of cloning the nucleic acid of the cap gene(s)of the virion.

In a preferred embodiment, the method for selection of a recombinantvirion invention further includes a step for the additional selection ofvirions, preferably an affinity binding step of virions (e.g. to a knownreceptor or binding motif that may be coupled to beads or a resin, forexample by an affinity chromatography), an ion exchange chromatographystep (to improve purifaction capabilites of such virions) or animmuno-selection step (to circumvent potential immune reactions frompatients, e.g. by immuno depletion with antibodies).

In a further preferred embodiment, the invention relates to a method forthe selection of a receptor binding motif comprising the steps asdefined above, wherein such second cell is permissive for the respectivevector.

Such receptor may be expressed recombinantly, preferably over-expressedby known recombinant technologies.

A further embodiment of the invention is a method for the in vivoselection of a recombinant virion capable of infecting a specific celltype comprising the steps of

-   -   (i) providing at least one first cell with a vector construct        comprising at least one nucleic acid from the previously        described library together with packaging sequences such as AAV        ITRs and one or more genes providing necessary non-structural        functions such as replication and packaging, for example of an        AAV Rep protein for the packaging of a virion;    -   (ii) providing such first cell with necessary cellular or viral        helper functions for the packaging of a virion if necessary;    -   (iii) incubating such first cell under suitable conditions for        the packaging of virions, preferably AAV, and collecting        produced virions, preferably AAV, by such first cell;    -   (iv) infecting an animal with such virions.

This method can be used for the identification of a mutant cap geneleading to virions having an increased infectivity or specificity for aspecific cell type by addition of the step of cloning the nucleic acidof the cap gene(s) from such cell type of the animal.

A further embodiment of this invention is a method for the selection ofa recombinant virion with a modified immunogenicity comprising the stepsof

-   -   i) providing at least one first cell with a vector construct        comprising at least one nucleic acid from the library of the        invention together with a second nucleic acid (especially        sequences such as AAV ITRs and one or more genes providing        non-structural functions such as replication and packaging, for        example functions of an AAV Rep protein) necessary for the        packaging of virion.    -   ii) providing such first cell with necessary cellular, viral,        physical and/or chemical helper functions for the packaging of        virions if necessary;    -   iii) incubating such first cell under suitable conditions for        the packaging of virions and collecting produced virions by such        first cell;    -   iv) applying an immunoselection step to the produced virions;    -   v) infecting at least one second cell with such collected        virions;    -   vi) providing such second cell with necessary cellular, viral,        physical and/or chemical helper functions for the packaging of a        virion;    -   vii) incubating such second cell under suitable conditions for        the packaging of virions and collecting produced virions by such        first or second cell;        whereas steps iv) to vii) can be repeated several times.

The non-structural functions as for example the Rep protein can beprovided in cis or in trans.

Furthermore, the first and the second cell can be of the same kind ortype.

The immunoselection steps are well known in the art. One can think ofvarious methods to use antibodies or similar molecules such as FABfragments or single chain antibodies to inhibit binding or uptake ofvirions by the second cell. For example one can pre-incubate theproduced virions with monoclonal or polyclonal antibodies. If antibodiesbind to a critical site of the virion that is involved in the mechanismof infection, this will result in a negative selection for virionsrecognized by such antibody. One could use either known monoclonalantibodies or sera from immunopositive mammals, especially humans. Thepolyclonal antibodies contained in this sera would have the advantage,that one can negatively select for virions that escape neutralizingantibodies without having the antibody isolated. A furtherimmunoselection step of choice is an immunodepletion reaction usingaffinity chromatography with antibody columns. Column material such asCNBr-activated Sepharose can be used to bind monoclonal or polyclonalantibodies. Produced virions can then be incubated with such antibodycolumn leading to an eluate of the column where binding virions havebeen depleted.

The invention further relates to a polypeptide comprising a peptide withthe sequence RGDAVGV, RGDTPTS, GKLFVDR, RDNAVVP, GENQARS, RSNGVVP,RSNAVVP or NSVRAPP.

In a preferred embodiment, the polypeptide of the invention consists ofa peptide with the sequence RGDAVGV, RGDTPTS, GKLFVDR, RDNAVVP, GENQARS,RSNGVVP, RSNAVVP or NSVRAPP.

According to a further preferred embodiment, the polypeptide of theinvention is a Cap polypeptide, preferably derived from a parvovirus,especially from an AAV.

The invention further relates to the use of a polypeptide as definedabove or comprising or consisting of a peptide with the sequenceRGDXXXX, RGDXPXX, or DDDXPXX with the exception of AGTFALRGDNPQG, forthe retargeting of eukaryotic viruses, preferably parvorviruses,especially AAV.

Furthermore, all identified peptides can be used for the targeting ofnon-viral vectors. Other potential uses of the peptides are triggeringor blocking cellular pathways e.g. by the activation or inhibition ofthe receptor due to the binding of isolated peptides to the respectivereceptor. The peptides also can be used as fusions with other peptidesor any other suitable molecules of choice. The peptides in this settingcan be used to couple such fusion to the surface of a cell for thepurpose of—for example—staining, tagging, sorting or killing of thecell.

The peptides can also be used for the purification of fusions or virionscontaining them by coupling the respective receptor onto beads andallowing binding of such fuions/virions to such coupled beads (affinitychromatography). Therefore such selected virions not only have theadvantage of a changed cell specificity but also that they can bepurified by affinity chromatography using their specific receptor.

The peptides RGDAVGV and RGDTPTS as well as RGDXXXX, RGDXPXX and DDDXPXXare useful in combination with cells that express RGD bindingintergrins. RGD binding integrins are receptors that are widelyexpressed among eukaryotic cells (Ruoslahti E (1996) Annual Review ofCell and Developmental Biology 12, 697-715; Aumailley M et al. (1990):FEBS Lett 12:262(1):82-6). An example for an integrin that binds RGDmotifs are the α_(v)β₅ and the α_(v)β₁ integrins. Such cells are forexample megakaryocytes, e.g. the cell line used for the screening M-07e.

The peptides GKLFVDR, RDNAVVP, GENQARS, RSNGVVP, RSNAVVP or NSVRAPP areuseful for B-CLL cells and Mecd cells. These peptides bind to one ormore cellular receptors that have not been identified so far. Every cellor cell line that expresses one or more of these receptors is apotential target for these peptides. It is known in the art how to testa cell or cell line, if one of the peptides is capable of binding to thecell surface. Since these peptides were identified by screening thelibrary against hematopoetic cells, it is reasonable to predict thatmany other hematopoetic cells will bind those peptides, for example Bcells.

The invention further relates to a recombinant virion obtainable by themethods of the invention for the selection of a recombinant virion.

Furthermore, the invention relates to a mutant cap gene obtainable bythe methods of the invention for the identification of a mutant capgene.

Furthermore, the invention relates to a Cap protein encoded by themutant cap gene of the invention.

Furthermore, the invention relates to a virion comprising the Capprotein of the invention.

Furthermore, the invention relates to a medicament for the treatment ofa patient suffering from cancer, an autoimmune disesase, an infectiousdisease or a genetic defect comprising a virion, a cap gene or a Capprotein of the invention.

Furthermore, the invention relates to a method for treating a patientsuffering from cancer, an autoimmune disesase, an infectious disease ora genetic defect comprising administering to the patient a virion, a capgene, or a Cap protein of the invention.

In this document, the content of all cited documents is included byreference.

The following examples and figures are intended to explain the inventionin detail without restricting it.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 Schematic map of the plasmid pwt99oen.

FIG. 2 Construction of the library of AAV-2 capsid modified particles. Apool of randomly generated oligonucleotides was cloned in an AAV-2genome encoding plasmid at the site corresponding to amino acidic site587 of capsid protein VP1. The obtained pool of plasmids was transfectedinto 293 cells. Following a standard virus production protocol, alibrary of approximately 10⁸ capsid modified AAV-2 clones was generated.

FIG. 3 AAV display screening procedure for the selection of retargetedmutants. Target cells were infected with the library of capsid modifiedAAV-2 clones and with adenovirus (helper for AAV replication). Noninfectious virions are removed by washing steps 2h post infection. Theviral progeny collected 48h p.i. was used for the next selection round.The evolution of the AAV population after each round was monitored bytiter determination and sequencing.

FIG. 4 Example of evolution of the viral population during 6 selectionrounds on M07e cells. (A) Dot blot assay quantification of viral progenyharvested after each infection cycle. (B) Sequencing of the randominsertion containing region of the cap gene shows the progressive lossof heterogenity in the viral population collected after each selectionround. After 5 rounds a single clone (in the shown example carrying aRGDAVGV inserted sequence) could be detected in the viral progeny.

FIG. 5 Transduction efficiencies Transduction efficiencies±standarddeviation as determined by FACS analysis in duplicate experiments forselected rAAV-GFP mutants (black bars). Transduction rates were alsoassessed after pre-incubation of viral preparation with soluble heparin(white bars) or pre-incubation of the cells with competing GRGDTP (graybars) and inactive GRGES peptides (checked bars).

-   -   a) M-07e cells.    -   b) Concentration dependece of RGDTP mediated inhibition of M-07e        cells transduction by rAAV/M07A (white circles) and rAAV-M07T        (black circles).    -   c) CO-115 cells.    -   d) HeLa cells.    -   e) Mec1 cells.    -   f) Primary B-CLL cells obtained from four different patients.

FIG. 6 A neutralization assay on HeLa cells. (A) Neutralizing antibodytiters against rAAV and rAAV-5 87/L1 4. Serial dilutions (1:10-1:1200)of 15 neutralizing human serum samples (P3- P65) were analyzed on HeLacells. As control, rabbit serum directed against the inserted L14-ligand(α-L14) was tested. The neutralizing titers (N₅₀) are expressed as thedilution at which transduction was 50% reduced compared to the positivecontrol. rAAV (B) and rAAV-587/L14 (C) were incubated with serum P35(1:80) prior infection of HeLa cells. GFP expression was monitored byfluorescence microscopy 48 hours post infection.

FIG. 7 A neutralization assay on B16FIO cells. Infection of irradiatedB16F10 cells with rAAV-587/L14 alone (A) or after co-incubation with P35serum (C) or anti-L14 serum (D) at a 1:80 serum dilution. Cells wereanalyzed for GFP expression by fluorescence microscopy after 72 hours.

FIG. 8 The effect of neutralizing antisera on rAAV-587/MecAtransduction. (A) After infection with adenovirus, Mec1 cells wereinfected with rAAV (top row) and rAAV-587/MecA (bottom row) alone(positive control) or after co-incubation with serum P35 at a 1:80dilution (+serum P35). Note that more physical particles were used forrAAV to achieve similar transduction. (B) FACS analysis of rAAV (toprow) and rAAV-587/MecA (bottom row) incubated with serum P35 (grey line)in comparison to their positive controls (black line). GFP expressionwas determined 48 hours post infection.

Tab. 1 Characterization and specificity of rAAV-GFP mutants with capsidmodifications. Genomic titers were measured by dot blot assay.Infectivity of the mutants for Hela, M07e and Mec1 cells was measured byFACS analysis after infecting the cells with identical genomicparticles/cell ratios. For each cell line, the transduction rate wasnormalized to 100% for the mutant corresponding to bold values. Theability of soluble heparin to inhibit infection of Hela cells wasassessed preincubating viral preparations with soluble heparin.

EXAMPLES Example 1 Methods

Production of Plasmids and Viruses

For the construction of plasmid pWt.oen, the HCMV promoter/enhancercassette and the GFP open reading frame in the plasmid pEGFPC-1(Clontech, Palo Alto, Calif.) were substituted with the wt AAV-2 genomeencoding fragment of plasmid pUC-AV2 (Girod A et al (1999) supra). A DNAfragment encoding amino acids AAAstopA and the restriction sites Notland Ascl was inserted between amino acid position 587 and 588 by PCRmutagenesis. To generate a library of AAV plasmids (p587Lib7) a pool ofsingle strand DNA molecules was synthesized as5′-TTGGCGCGCCGCVNNVNNVNNVNNVNNVNNVNNGGCGGCCGCTTTTTTCCTTGA-3′(whereas N=A/G/C/T, V=A/GIG/C (not T))and HPLC purified (Metabion GmbH, Martinsried, Germany). For thesynthesis of double-stranded molecules a 5′-CTCAAGGAAAAAAGC-3′ primerwas used. dsDNA molecules were cloned into the Ascl-Notl large fragmentof plasmid pWt.oen, p587Lib7 was electroporated into E. coli strain DH5ausing a Gene Pulser (Biorad, Hercules, Calif.) and amplified DNA waspurified. The efficiency of the transformation was controlled by platingsample aliquots. DNA of more than 20 clones was controlled by sequencingwith the primer 4066Back (5′-ATGTCCGTCCGTGTGTGG-3′). Plasmids pRC, pXX6(obtained from J. Samulski, Chapel Hill, N.C.) and psub/CEP4/EGFP werepreviously described (Girod A et al (1999) supra, Xiao X et al (1998) J.Virol. 72, 2224-32).

For the production of viruses, 15 150 mm Petri-dishes of 293 cells at80% confluence were co-transfected with 37.5 μg of DNA. For theproduction of the AAV library, p587Lib7 and plasmid pXX6 wereco-transfected at a molar ratio of 1:1. For the production of rAAV-wt,the cells were co-transfected with vector plasmid psub/CEP4/EGFP,packaging plasmid pRC and adenoviral plasmid pXX6 at a molar ratio of1:1:1. For the production of the capsid modified GFP expressing rAAVmutants, pRC plasmids modified to contain the appropriate Notl-Asclretargeting insertion were used. L14-AAV was produced using plasmidpI-587 instead of pRC (Girod A. et al supra). After 48 hrs cells werecollected and pelleted by centrifugation. Cells were resuspended in 150mM NaCl, 50 mM Tris-HCl (pH 8.5), freeze-thawed several times, andtreated with Benzonase (50 U/ml) for 30 min at 37° C. Cell debris wasremoved by centrifugation, supernatant was loaded onto an iodixanolgradient and subjected to 69000 rpm for 1 hr at 18° C. as described(Zolotukhin, S et al. (1999) Gene Ther. 6, 973-85). Virions were thenharvested from the 40% iodixanol phase and titrated by DNA dot-blothybridization (Girod A. et al., supra).

Tissue Culture

HeLa cells (human cervix epitheloid carcinoma, ATCC CCL 2), M-07e cells,a human megakaryocytic leukemia cell line (obtained from James D.Griffin, Boston, Massachussets), Mec1, a cell line derived from apatient with B-CLL in prolymphocytoid transformation (obtained fromFederico Caligaris-Cappio, Torino, Italy), CO-115 cells (human coloncarcinoma), and 293 cells (human embryonal kidney) were maintained inDulbecco's modified Eagle's medium (DMEM) (HeLa and 293),DMEM/NUT.Mix.F-12 medium (CO-1 15), RPMI medium (M-07e) or Isocove'smedium (Mec1) supplemented with 10% fetal calf serum (FCS), penicillin(100 U/ml) and streptomycin (100 μg/ml), and L-glutamine (2 mM), at 37 °C. and 5% CO₂. For M-07e cells, 10 ng/ml interleukin 3 (IL-3) was addedto the medium.

Peripheral blood was obtained with informed consent from four patientswith an established diagnosis of B-CLL. Mononuclear cells were isolatedon a Ficoll/Hypaque (Seromed, Berlin, Germany) density gradient bycentrifugation, depleted of monocytes by adherence to plastic tissueculture flasks and cultivated in Isocove's medium supplemented as forMec1 cells. More than 98% of isolated cells co-expressed CD5 and CD19 asassessed by flow cytometry, therefore non-malignant B cells did notconstitute a meaningful fraction of the total cells isolated. Patientswere either untreated or had not received cytoreductive treatment for aperiod of at least one month before investigation and were clinicallystable and free from infectious complications.

Determination of Transduction Efficiencies

Cells were seeded in 96 or 24 well plates (Nunc, Wiesbaden, Germany) andinfected with rAAV-GFP clones, harvested 48 hrs p.i., washed andresuspended in 1 ml PBS. The percentage of GFP expressing cells wasdetermined by flow cytometry with a Coulter Epics XL-MCL (BeckmanCoulter, Krefeld, Germany). A minimum of 5000 cells were analyzed foreach sample. Infectivity of the retargeted mutants was determined in thepresence or absence of various concentrations of GRGDTP or GRGESpeptides (Bachem, Bubendorf, Swiss) or 5 I.U./[II soluble heparin(Braun, Melsungen, Germany).

Selection of AAV-2 Retargeted Mutants

10⁷ target cells were super-infected with 1000 genomic libraryparticles/cell and with adenovirus at an MOI of 20 and incubated at 37°C. 2 hrs p.i. cells were centrifuged, resuspended in fresh culturemedium and incubated at 37° C. 48 hrs p.i., cells were rinsed with 5 mlPBS, resuspended in 5 ml of lysis buffer (150 mM NaCI, 50 mM Tris/HCl,pH 8.5) and lysed through 3 freeze/thaw cycles. Cellular debris wasremoved by centrifugation and the supernatant was used to infect thenext batch of target cells (second round of infection). After eachselection round viral DNA was purified from a 100 μl aliquot of thecrude lysates by phenol/chloroform extraction and the 587 region wassequenced (primer 4066-back).

Example 2 Selection of AAV-2 Retargeted Mutants for M-07e and Mec1 Cells

We generated a library of 4×10⁶ capsid modified viral particles carryingrandom insertions of 7 amino acids at the position 587 (FIG. 2 andExample 1). The pool of capsid mutants was subjected to repeated cyclesof infection and harvesting of the viral progeny from the target cells(FIG. 3). Virions with impaired ability to enter the cells were removedby changing the culture medium 2 hrs post infection (p.i.). Viralprogeny was extracted from the cells 48 hrs p.i. by freeze/thaw cyclesand used to infect a new batch of target cells in a new selection round.After each harvest, a small aliquot (100/tl) of the crude lysate wasused to extract viral DNA. By titrating this DNA and sequencing the 587region it was possible to monitor the evolution of the library (FIG. 4).The selective pressure provided by the culture environment drove theselection by means of their ability to accomplish every step in theinfection process, namely binding, uptake, uncoating, nucleartranslocation, replication and gene expression.

The potential of the AAV display system for the generation of retargetedmutants was tested on two cell lines that are resistant to wt AAV-2infection.

M-07e is a human megakaryocytic cell line (Avanzi G C et al (1988) Br.J. Haematol. 69, 359-66). Failure of AAV-2 to infect these cells hasjustified the use of this cell line as negative control in severalreported AAV-2 infection experiments Bartlett J S et al. (1999) Nat.Biotechnol. 17, 181-186; Ponnazhagan S et al (1996) J. Gen. Virol. 77,1111-22).

Mec1 is a cell line derived from B-cell chronic lymphocytic leukemia(B-CLL) cells in prolymphoid transformation (Stacchini A et al. (1999)Leuk. Res. 23, 127-36) and is also resistant to wt AAV-2 infection.

A typical selection is depicted in FIG. 4. The amount of viral DNAdetected in the crude lysates and the analysis of the sequence showedthat the number of recovered virions increased after each round, whilethe heterogeneity of the pool was progressively lost. After 5 roundsonly one single clone was present in the viral progeny. Application ofthe library to M-07e cells led to the selection of a clone carrying anRGDAVGV sequence at the 587 site (FIG. 4). In a parallel experiment weisolated a clone which carried an RGDTPTS sequence. Interestingly, bothclones isolated from M-07e cells led to the selection of an RGD motif,known to bind to several types of cellular integrins (Ruoslahti E (1996)Annu. Rev. Cell. Dev. Biol 12, 697-715). Analogous experiments performedwith Mec1 cells led to the identification of clones carrying GENOARS,GKLFVDR, NSVRAPP and RSNAVVP/RSNGVVP peptides, respectively (data notshown).

Example 3 Cloning of Selected Mutants

The selected DNA sequences were cloned into appropriate plasmids for theproduction of capsid-modified recombinant AAV (rAAV) vectors encodingthe enhanced Green Fluorescent Protein (GFP). CorrespondingGFP-expressing retargeted vectors rAAV-M07A (RGDAVGV insertion),rAAV-M07T (RGDTPTS insertion), rAAV-MecA (GENQARS insertion) andrAAV-MecB (RSNAVVP insertion) were produced (see Example 1) and genomictiters were determined by dot blot assay. Genomic titers of the selectedmutants were comparable or higher than titers of AAV vectors withunmodified capsid (rAAV-wt) (Tab. 1).

Example 4 Transduction Efficiencies of Retargeted Vectors

The selected capsid mutants were tested for their ability to transduceM-07e cells (FIG. 5 a). At a genomic particle/cell ratio of 2×10⁴, themutants rAAV-M07A and rAAV-M07T transduced 50±2.5% and 47±2.7% of M-07ecells, respectively, representing a 100 and 94 fold increase incomparison to rAAV-wt transduction efficiency (0.5±0.01%). In contrast,rAAV-MecA and rAAV-MecB transduced M-07e cells with an efficiency ofonly 8.1±1.5% and 16±2%. The vector rAAV-L14, carrying an RGD motifinserted at position 587 (Girod A et al (1999) supra), was alsocompared. Interestingly, rAAV-L14 transduced only 10±0.7% of M-07ecells, which was five times less efficient than the selected mutantsrAAV-M07A and rAAV-M07T. This highlighted the advantage of thecombinatorial approach when compared with the simple insertion of anexogenous sequence.

Example 5 Tropism of Retargeted Vectors

We then examined whether the transduction of M-07e cells by rAAV-M07Aand rAAV-M07T vectors was specifically mediated by the amino acidsinserted at position 587. In the capsid of wt AAV, the region aroundposition 587 is involved in the binding to heparan sulfate proteoglycan(HSPG) (Nicklin S A et al. (2001) Mol. Ther. 4, 174-81; Wu P. et al.(2000) J. Virol. 74, 8635-47), the primary receptor of AAV-2 (SummerfordC and Samulski J (1998) J. Virol. 72, 1438-45). Pre-incubation withsoluble heparin, an HSPG analogue and competitor, inhibited transductionof M-07e cells by rAAV-MecB but not by rAAV-M07A, rAAV-M07T andrAAV-MecA (FIG. 5 a). This indicated that the insertion of appropriateheterologous amino acids at this site abolished the requirement of AAVto use HSPG as a primary receptor for transmembrane entry. In markedcontrast, preincubation of M-07e cells with a competing soluble GRGDTPpeptide (450 μM) almost completely inhibited transduction of M-07e cellsby rAAV-M07A and rAAV-M07T (FIG. 5 a). This effect wasconcentration-dependent (FIG. 5 b). Pre-incubation with an inactive(GRGES) peptide (450 μM) had no effect (FIG. 5 a). Taken together, theresults demonstrate that rAAV-M07A and rAAV-M07T transduce target cellsthrough the specific interaction of the selected RGD motif presented onthe viral capsid with an integrin receptor expressed on the surface ofthe target cells.

We also examined the selected mutants on cells which expressed HSPG andwere permissive for wt AAV-2 infection. In human colon carcinoma CO-115cells (Carrel S et al. (1976) Cancer Res. 36, 3978-84) the transductionefficiency of the virus mutants rAAV-M07A, rAAV-M07T, rAAV-MecA andrAAV-MecB was reduced by 50, 43, 12 and 31%, respectively, when comparedto wt AAV-2 (FIG. 5 c), while it was similar to wt AAV-2 in HeLa cells(FIG. 5 d). In both cell lines, transduction by mutants rAAV-M07A andrAAV-M07T was blocked almost completely by the GRGDTP peptide, but notby the GRGES peptide nor by heparin. In contrast, transduction byrAAV-wt and rAAV-MecB was inhibited by heparin but not by the GRGDTPpeptide (FIG. 5 c and d). Moreover, cells which lacked the expression ofan integrin receptor were not permissive for transduction by the mutantsrAAV-M07A and rAAV-M07T (data not shown). Taken together, these resultsdemonstrate that the integrin receptor recognizing the RGD peptide onrAAV-M07A and rAAV-M07T capsids is also expressed on CO-115 and HeLacells. Therefore, the tropism of the selected capsid mutants is notrestricted to hematopoietic cell lines, but to an integrin receptor,which is probably widely expressed.

Successful retargeting of mutants selected on Mec1 cells is depicted inFIG. 5 e. While transduction of Mec1 cells by rAAV-wt was notdetectable, mutants rAAV-MecA and rAAV-MecB transduced up to 23% ofthese cells at a genomic particle/cell ratio of 4×10⁴. Using rAAV-MecA,we then examined the transduction efficiency in primary leukemia cellsin order to explore the potential clinical relevance of the AAV displaytechnology. Primary B-CLL cells are resistant to transduction by mostcurrently available vital vector systems, including AAV (Cantwelf M J etal (1996) Blood 88, 4676-83; Rohr U P et al (1999) Blood 94, 181a). Inremarkable contrast to vectors with unmodified capsid, rAAV-MecA (8×10⁴genomic particles/cell) transduced primary leukemia cells isolated fromfour B-CLL patients at an efficiency of 54, 49, 23 and 21%, respectively(FIG. 5 f). In contrast, rAAV-M07A and rAAV-M07T failed to transduceprimary B-CLL cells (data not shown). The results indicate that suchmodified vectors might be useful for an AAV-based gene therapy of B-CLLCantwell M et al. (1997) Nature Med. 3, 984-89; Wierda W G et al. (2000)Blood 96, 2917-24).

Any successful attempt to molecularly engineer viruses for human somaticgene therapy will depend on our ability to generate retargeting vectorsthat retain the major functions required for appropriate intracellularprocessing. Our findings seem highly relevant in this regard. Because ofthe complexity of the virus-cell interaction, it is highly advantageousto screen appropriate virus mutants from a large library rather than togenerate a limited number of virus variants by a more or less educatedguess. Since no refinement of the selection process was under-taken,some limitations remained: the capsid mutants showed receptorspecificity, but not cell specificity. However, the goal of producingviral clones with a further restriction of the virus tropism should beachieved by adding steps to the screening process which deplete thoseclones able to infect undesirable cell types. An additional upgrade ofthis technology might be the generation of an AAV library withrandomized insertions in multiple sites of the capsid. Moreover, thevirus display might be also used for the identification of capsidvariants that are less efficiently recognized by human antibodies orimmune effector cells. Finally, the shortness of the insertions thatwere successfully used to generate retargeting clones suggests that thistechnology might be applicable in other viral systems.

Example 6

In two completely independent experiments, after 5 selection rounds onM07e cells (resistant to wt AAV-2 infection) the sequences obtainedshowed no more randomized features at the site of the insertion and wewere able to characterize two highly homologue RGD motifs containingsequences: RGDAVGV and RGDTPTS. Oligonucleotides encoding for thesepeptidic sequences were cloned into GFP-AAV plasmids. The correspondentmutants were packaged and used to infect MO7e cells. For both mutants,infection of M07e cells with 2000 genomic particles/cell resulted intransduction rates higher than 86% (wt AAV-2 transduction rate was lessthan 6%).

Selection rounds performed on a cell line (Mec1) derived from B-CLLcells in prolymphoid transformation, led to the identification ofseveral sequences that provided AAV capsids with improved infectionefficiency on these cell types. In particular the sequence GENQARSconferred to GFP-AAV virions transduction rates of up to 20% on Meclcells, and to 55% on primary B-CLL cells (both cell types arenon-permissive to wt AAV infection).

Production of the AAV Library.

The cloning strategy is depicted in FIG. 2. A combinatorial library ofAAV for the selection of retargeted clones was generated by cloningrandomly generated oligonucleotides with a length of 21 bases at thegenomic site corresponding to aa position 587 using the plasmid pWT99oen(FIG. 1, sequence given).

The inserted sequence consisted of 7 repetitions of NNB codons (N=A,C,G,or T; B=C,G or T) to allow a 50% reduction of stop codons probability.

While the wild type aminoacids flanking the 587 position were allretained, a triple and a double Ala sequence was engineered upstream anddownstream of the randomized sequence, respectively, in order toincrease the flexibility of the inserted peptide and to reduceconformational stress of the native capsid structure.

We obtained approximately 5×10⁷ plasmids containing the randomlygenerated insertions. More than 20 of these plasmids were sequenced tocontrol the outcome of the cloning; all the sequenced plasmids containeda randomized 21 bases insertion in the correct position.

This pool of plasmids was transfected into 293 packaging cellsconcomitantly to a helper plasmid containing the genes of adenovirus,necessary for the packaging of AAV virions.

Viral progeny was harvested by a standard purification protocol on aniodixanol discontinuous gradient (Samulski et al.).

Genomic and infectious titers of the viral preparation were measured bydot blot and immunofluorescence analysis using an anti rep antibody andquantified in respectively 4×10¹¹ virions/ml and 6×10⁸/ml.

The sequence obtained after digestion of viral proteins with ProteinaseKand phenol/chloroform extraction is depicted in FIG. 4 and confirms therandomized nature of the insertion at the 587 site.

Selection of Efficiently Infecting Mutants on Target Cells.

To demonstrate the feasibility of the combinatorial selection approach,we performed several infection and harvest rounds of the AAV library onMo7e and Mec1 cells (both cell lines being almost completely resistantto wild type AAV infection) in order to isolate the clones with betterinfection ability. This was simply achieved by performing repeatedinfection/harvesting cycles on the target cells. A schematicrepresentation of the procedure is depicted in FIG. 3. Adenovirus at aMOI of 100 was used as helper for the replication of AAV.

In this system, the cultural environment exerts a strong selectivepressure contemporarily on binding, entry, replication and packagingability of the viral clones. Viral replication itself exerts in theinfected cells the amplification step necessary to augment the number ofviable mutants that will be harvested and used for the subsequentselection rounds.

2 hours p.i., the culture medium was changed to remove non-infectiousmutants. 48 hours p.i. cells were centrifuged, rinsed with PBS,resuspended in 5 ml lysis buffer and subjected to 3 freeze/thaw cyclesto allow diffusion of the progeny virions into the solution. Cellulardebris was separated by centrifugation at 5000 g.

After each infection/harvesting round, a small aliquot of the crudelysate preparation was used to measure the genomic titer of thepreparation and for sequencing of the respective viral population. Theremaining preparation was used to infect the next batch of target cells.

Identification and Characterization of Mutants Retargeted to M07e Cells

The M07e cell line is resistant to wt AAV-2 infection. Thischaracteristic has been attributed to the lack of expression of theputative primary receptor for AAV-2 (heparan sulfate proteoglycan), andhas justified the use of this cell line as negative control in manyreported AAV-2 infection experiments.

FIG. 4 shows the results of 5 rounds of infection/harvesting of the AAVpool on this target cells. Round after round, we could observe a slightincrease of the AAV genomic titer in the crude lysates preparation (asassessed by dot blot analysis). Concomitantly, the peaks of thesequence-reaction chart in the random insertion portion becameincreasingly higher during the selection procedure, and at the 5^(th)cycle it was possible to read a fixed sequence from the sequencingreaction.

The selection procedure was performed in two independent experiments andresulted in the identification of two DNA sequences encoding forrespectively RGDAVGV and RGDTPTS peptides. FIG. 4 depicts only theexperiment that generated the RGDAVGV sequence.

The selected DNA sequences were cloned into appropriate plasmids for theproduction of capsid-modified recombinant AAV vectors encoding for theEnhanced Green Fluoresent Protein (rAAV-GFP). GFP expressing versions ofthese retargeted clones (rAAV-M07A containing the RGDAVGV sequence andrAAV-M07T containing the RGDTPTS sequence) were produced by standardrAAV production protocols.

The ability of mutants rAAV-M07A and rAAV-M07T to transduce M07e cellswas compared with the efficiencies of vectors with unmodified capsid(rAAV-wt) and of vectors expressing the L14 sequence at the 587 site(rAAV-L14). M07e cells were infected with identical genomicparticles/cell ratios (FIG. 5). Transduction rates were higher than 88%when using the retargeted mutants, 6% using unmodified capsid mutantsand 18% using rAAV-L14.

Similarly to the selected mutants, rAAV-L14 carries a RGD motifcontaining sequence (of the laminin fragment P1) inserted at the 587site. The more than 5 fold higher efficiency of the mutants generated byour display system in comparison with rAAV-L14 clearly highlights theadvantages of the combinatorial display approach where the modificationsare selected for their efficiency directly in the vector contest, incomparison with the simple insertion of a previously known retargetingsequence.

To demonstrate the specificity and the receptor-mediated nature of theinfection process, we measured M07e cell transduction rates of the viralclones in the presence of a competing RGDS peptide or an inactive RGESpeptide. Incubation of target cells with 100 uM RGDS peptides prior toinfection reduced transduction efficiencies by more than 50%.Pre-incubation of the cells with the RGES peptide failed to inhibitinfection (FIG. 5A).

Identification and Characterization of B-CLL Cells Retargeted Mutants.

Mec1 is a cell line derived from B-Cell Chronic Lymphocytic Leukemiacells in prolymphoid transformation (Stacchini et al.) and is resistantto wt AAV-2 infection.

After 3 rounds of selection on Mec1 cells, it was possible to read aninserted GANGANNACNNNNCNANNANN nucleotidic sequence at the 587 site.

In other setups of the selection procedure on this type of cells, after5 rounds we could isolate viral clones with insertions encoding forpeptides GKLFVDR, GENQARS, RSNGVVP, or NSVRAPP.

The GENQARS sequence was cloned into an appropriate plasmid for theproduction of capsid-modified recombinant AAV vectors encoding for theEnhanced Green Fluoresent Protein (rAAV-GFP). GFP expressing viralparticles of this retargeted clone (rAAV-Mecl) were produced by standardrAAV production protocols.

Infection of Mec1 cells with rAAV-Mec1 (20000 genomic particles/cell)resulted in a transduction rate of approximately 20%, while rAAV-wtfailed to transduce more than 2% of these cells (FIG. 5B).

Primary B-CLL cells are resistant to transduction with most currentlyavailable viral vector systems and previous reports failed to show forAAV vectors transduction rates greater than 3% (citation from David'spaper). rAAV-Mec1 showed transduction efficiencies of 54, 49, 21 and 23%when applied to primary cells obtained from 4 B-CLL patients (FIG. 5C).With these transduction rates, an AAV-based gene therapy approach forthe cure of B-cell Chronic Lymphocytic Leukemia is now possible for thefirst time. Moreover, patient-specific differences in the permissivityof these primary cells to AAV vectors are suggested by these results andconfirmed by other data obtained (Wendtner et al. paper submitted). Thevirus-display technology opens the horizon for the generation of patientspecific vectors. Optimization of the protocol for the selection ofretargeted mutants directly on primary B-CLL cells shall lead to achievethis goal and efforts in this direction are currently spent in ourlaboratory.

Example 7

Transduction of HeLa Cells by rAAV-587/L14 is not Inhibited byPreexisting Neutralizing Antibodies in Human Serum Samples

A detailed understanding of major immunogenic domains on theadeno-associated virus (AAV) capsid is not only important with regard tothe binding of serum antibodies to the virus and its subsequentneutralization by the immune system, but also with regard to theexistence of neutralizing antibodies that directly inhibit infection ofthe target cells by AAV vectors. To analyze the interference ofdifferent human antisera with AAV transduction, we used a recombinantAAV vector coding for GFP and carrying the L14 ligand at position 587(rAAV-587/L14) to determine whether this modification would block theneutralizing ability of human antisera.

First, we determined the presence of neutralizing antibodies in humanserum samples. 43 serum samples positive for AAV antibodies (Ab) weretested in a neutralization assay with an AAV vector coding for GFP,which carried the wild-type AAV capsid (rAAV). rAAV was incubated withserial dilutions of serum samples prior to transduction of HeLa cells.Thereafter, the number of GFP expressing cells was assessed by FACSanalysis. Neutralizing titers were defined as the serum dilution wheretransduction was reduced by 50% (N₅₀). Serum samples were considered asneutralizing when the N₅₀ was 1:320 or higher. 31 of these 43 serumsamples (72%) contained neutralizing Ab against AAV, in agreement withpreviously published data (Erles K et al. (1999) J Med Virol 59:406-11).

15 of these 31 serum samples were randomly selected for furtheranalysis. The effect of these serum samples on the transduction of HeLacells by rAAV-587/L14 as compared with rAAV was determined (FIG. 6A). Inaddition, the neutralizing monoclonal Ab C37-B (Wobus C E et al. (2000)J Virol 74: 9281-93) and an anti-L14 serum (generated against the L14ligand) were tested. For these experiments identical transducingparticle numbers of rAAV-587/L14 and rAAV were used. Both vectors wereincubated with serial dilutions of neutralizing serum samples prior totransduction of HeLa cells. For all serum samples tested, transductionby rAAV-587/L14 was 8 up to 64 fold less reduced than transduction byrAAV (mean 15 fold). In 13 out of 15 serum samples, transduction byrAAV-587/L14 was only slightly impaired, with neutralizing titers of1:80 or lower, demonstrating the ability of rAAV-587/L14 to escape theeffects of neutralizing Ab (FIG. 6A). Strikingly, rAAV-587/L14 was ableto escape the neutralizing Ab in serum P47 at any dilution tested, andserum samples P17, P31 and P37 reduced transduction only at a dilutionof 1:20, where unspecific interactions could not be excluded. FIGS. 6Band 6C show one representative experiment with serum P35, whichcompletely inhibited transduction by rAAV at a 1:80 dilution (FIG. 6B).In marked contrast, transduction by rAAV-587/L14 was not affected (FIG.6C). Only two serum samples (P16 and P48) were able to neutralizerAAV-587/L14 transduction efficiently, with a N₅₀ of 1:320. We assumethat this was due to the high neutralizing Ab content in these serumsamples, because transduction by rAAV-587/L14 still remained lessaffected than transduction by rAAV. As an additional control, themonoclonal Ab C37-B was tested. C37-B is a neutralizing Ab that inhibitsbinding of AAV to the host cell (Wobus C E et al. supra). It failed tobind rAAV-587/L14 in an ELISA (data not shown), therefore it should notinterfere with rAAV-587/L14 transduction. As expected, rAAV-587/L14transduction was not neutralized by C37-B, while rAAV transduction couldbe totally inhibited by this antibody (data not shown). In markedcontrast, anti-L14 serum, which was generated against the L14 ligand,neutralized rAAV-587/L14 transduction completely at a 1:160 dilution,while rAAV transduction remained unaffected (FIG. 6A). To rule out thepossibility that these observations were due to different numbers ofphysical particles used for rAAV and rAAV-587/L14, we performedadditional control experiments, where neutralization assays wereperformed with identical numbers of physical particles for both AAVvectors. These experiments yielded identical results (data not shown).

Taken together, these results demonstrate that the mutant rAAV-587/L14is able to escape preexisting neutralizing Ab in human serum samples.

Neutralizing Sera Do Not Interfere with the L14 Mediated Tropism ofrAAV-587/L14 on B16F10 Cells

Insertion of the integrin specific L14 peptide in 587 expands thetropism of AAV to non-permissive B16F10 cells (Girod A et al. (1999) NatMed 5: 1052-6). To determine if rAAV-587/L14 was able to retain itsability to infect the target cell line B16F10 via the inserted ligandL14 in the presence of neutralizing antisera, we performed additionalexperiments with selected serum samples. rAAV-587/L14 was incubated withserial dilutions of P35 serum before transduction of irradiated B16F10cells. After 72 hours GFP expression was measured, rAAV-587/L14efficiently transduced B16F10 cells despite incubation with P35 at a1:80 dilution, whereas anti-L14 serum completely inhibited transductionat this dilution (FIG. 7B and 7C). When testing P37 and P26, the sameneutralizing titers as determined on HeLa cells were obtained (data notshown). These findings showed that the AAV L14 targeting vector couldescape neutralizing antibodies in human sera while retaining itsretargeting ability.

The Ability of rAAV-587 to Escape Neutralizing Sera Does Not Depend onthe inserted L14 Ligand

To exclude that the escape from neutralizing antisera was caused by aspecific ligand, we tested another insertion mutant, rAAV-587/MecA thatcarries a 7 aa ligand (GENQARS) at position 587. This mutant has beenselected by AAV-display on Mec1 cells and efficiently transduces Mec1cells and primary B-cells from chronic lymphocytic leukemia patients ina receptor specific manner (as described before). rAAV-587/MecA and rAAVwere incubated with the serum P35 before Mec1 cells were infected.Transduction of Mecl cells by rAAV-587/MecA was not affected by theneutralizing Ab of serum P35 (1:80 dilution). In contrast, rAAVtransduction was almost completely inhibited by this serum (FIG. 8).Experiments with other neutralizing serum samples provided identicalresults (data not shown). As with HeLa cells, A20 was able to inhibittransduction of rAAV-587/MecA, while C37-B had no effect (data notshown).

Taken together, the results demonstrate that the insertion of differentheterologous ligands at position 587 allows escape from preexistingneutralizing antibodies. Targeting properties of these vectors areretained in these capsid mutants, even in the presence of neutralizingantisera.

Discussion

This is the first description of the generation and application of aneukaryotic virus combinatorial library for the identification of celltype specific viral gene delivery vectors. In the described experiments,the application of this technique resulted in the description of severalnew AAV mutants with high efficiency of transduction of cells that areresistant to wt AAV infection.

All mutants described herein are specific, and show tropismcharacteristics determined by the insertion at the 587 site, asdemonstrated by the RGDS peptide competition experiments (FIG. 5A). Alsothese clones showed no interaction with the natural primary receptor ofAAV, heparan sulfate proteoglycan (Tab.1). Further specificity of thevirions could be achieved introducing other modifications of the capsidstructure, e.g. combining the insertion of retargeting sequences withmodifications such as 561-565 DEEEI-AAAAI substitution (Wu et al.),and/or the AISP insertion at nucleotidic site 3761 (Rabinowitz et al.).

Another possibility to increase specificity of the vectors is theintroduction of subtractive selection rounds, e.g. infecting cells forwhich the infection is undesired and recovering the non-infectiousvirions containing supernatant, or using affinity columns to deplete theviral population from column-binding clones.

A further upgrade of the system that is underway in our lab is thegeneration of an AAV library with randomized insertions at the level ofmultiple capsid protein sites.

The description of the mutant GENQARS, that showed transduction rates upto 55% of B-CLL primary cells, has immediate relevance for gene therapyof this malignancy. Previous attempts to transduce these cell type withAAV-based vectors failed to achieve efficiencies greater than 3%. Ourresults also suggest the importance of establishing protocols for thegeneration of patient-specific vectors. With the AAV Display technologythis is now possible.

The comparison of the efficiency of infection of the randomly selectedmutants with the L14 mutant, demonstrates the importance of the specificposition of the RGD sequence, and of the flanking amino acids andtherefore suggests the advantages of selecting the retargetingmodifications directly in the structural context of the vector.

The technology described herein for the adeno-associated virus can beadapted for any viral system.

Besides the goals of gene delivery systems, the AAV display will be avaluable tool for the understanding of the biology of this virus, forthe characterization of peptides with interesting biological properties,and for the investigation of specific ligand-receptor interactions. TAB.1 Infectivity Heparin Genomic Infectivity Infectivity on B-CLL Inhibi-Viral Clone Titer/ml on Hela on M07e cells tion wt 5 × 10¹⁰ 100%  1% 3% + L14 10¹⁰  2%  20% n.d. − rAAV-M07A 10¹¹ 108% 100%  10% − rAAV-M07T10¹¹  91%  84%  29% − rAAV-Mec1 5 × 10¹⁰ 100%  39% 100% −

1-76. (canceled)
 77. A method for the production of a library of nucleicacids comprising a multiplicity of expressible structural genes from atleast one eukaryotic virus, comprising the steps of: a) providing a setof nucleic acids, each encoding at least one structural gene from aeukaryotic virus and comprising a suitable packaging sequence, and b)inserting a first insert (1) into the structural gene.
 78. The method ofclaim 77, wherein the structural genes are from an enveloped virus suchas a retrovirus, lentivirus, or herpes virus, e.g. HSV1, HSV2, EBV,Varizella zoster virus, human herpes virus 1, 2, 3, 4, 7 or
 8. 79. Themethod of claim 77, wherein the structural genes are cap genes from anon-enveloped virus such as parvovirus or adenovirus.
 80. The method ofclaim 79, wherein the cap genes are from a parvovirus selected from thegroup consisting of Adeno-associated Virus (AAV), Canine Parvovirus(CPV), MVM, B19, H1, AAAV, and GPV.
 81. The method of claim 80, whereinthe cap genes are from an AAV.
 82. The method of claim 77, wherein theset of nucleic acids is derived from one nucleic acid.
 83. The methodaccording to claim 77, wherein by inserting insert (1) a sequence of thestructural gene is removed.
 84. The method according to claim 83,wherein the removed sequence comprises or is part of an insert (2)inserted into the structural gene before step (a).
 85. The methodaccording to claim 84, wherein insert (2) prevents the formation of afunctional capsid protein, preferably by containing a stop codon. 86.The method according to claim 85, wherein by inserting insert (1) thestop codon is removed.
 87. The method according to claim 77, wherein thenumber of nucleotides of insert (1) is three or a multiple of three. 88.The method of claim 84, wherein the number of nucleotides of insert (2)is three or a multiple of three.
 89. The method of claim 77, whereininsert (1) is inserted at a region of the cap gene encoding amino acidson the surface of the structural protein.
 90. The method of claim 89,wherein the virus is AAV and wherein the insert (1) is inserted after anucleic acid corresponding to any site within the first amino terminalamino acids 1 to 50, or corresponding to amino acid positions 261, 381,447, 534, 573, and/or 587 of the capsid protein VP1, preferably to aminoacid position 447 or
 587. 91. The method of claim 77, wherein insert (1)is randomly or partially randomly generated.
 92. The method of claim 77,wherein insert (1) does not contain any stop codons.
 93. The method ofclaim 77, wherein the library has a multiplicity of viral mutants thatis greater than 10², preferably greater than 10⁵, especially greaterthan 10⁶.
 94. A library of nucleic acids comprising a multiplicity ofexpressible structural genes, preferably cap genes from a parvovirus,more preferably from an Adeno-associated Virus (AAV), an CanineParvovirus (CPV), MVM, B19, H1, AAAV or GPV.
 95. The library accordingto claim 94, wherein the multiplicity of expressible structural genes,preferably cap genes is greater than 10², preferably greater that 10⁵,especially greater that 10⁶.
 96. The library according to claim 94,wherein the library has a multiplicity of viral mutants that is greaterthan 10², preferably greater than 10⁵, especially greater than 10⁶. 97.The library according to claim 94, wherein the nucleic acid is a linearnucleic acid, a plasmid, a viral particle or a viral vector, e.g. arecombinant AAV, Adenovirus or Herpes Simplex Virus vector.
 98. Thelibrary according to claim 94, wherein the nucleic acid furthercomprises packaging sequences such as AAV ITRs and at least oneexpressible gene providing necessary functions for replication andpackaging of virions such as an AAV Rep protein.
 99. The libraryaccording to claim 94, wherein the nucleic acid is DNA.
 100. The libraryaccording to claim 94, wherein the cap gene is derived from one of theAAV serotypes from the group comprising AAV1, AAV2, AAV3, AAV4, AAV5 andAAV6.
 101. The library according to claim 94, wherein the cap gene isderived from the cap gene encoded in plasmid pWT99oen.
 102. The libraryaccording to claim 94, wherein a multiplicity of nucleic acid sequencesare inserted into at least one site of the structural gene, preferablyof the cap gene, wherein the number of inserted nucleotides is three ora multiple of three.
 103. The library according to claim 102, whereinthe inserted nucleic acid sequences are randomly generated, especiallyusing NNN codons, NNB codons or NNK codons.
 104. The library accordingto claim 102, wherein the inserted nucleic acid sequences are partiallyrandomly generated, especially using codons with one, two or three fixednucleotides.
 105. The library according to claim 102, wherein theinserted nucleic acid sequences have a length of at least 3 nucleotides,preferably of at least 9, especially at least 18 nucleotides.
 106. Thelibrary according to claim 102, wherein the inserted nucleic acidsequences were inserted using standard restriction endonucleases orrecombination systems, preferably the gateway or the cre/loxrecombination system or polymerase chain reaction techniques, preferablyusing degenerated primers.
 107. The library according to claim 102,wherein the inserted nucleic acid sequences lead to an insertion ofamino acids into the VP1, VP2 and/or VP3 structural protein, preferablyat a site that is located on the surface of the capsid.
 108. The libraryaccording to claim 102, wherein the inserted nucleic acid sequences areinserted after a nucleic acid corresponding to any site within the firstamino terminal amino acids 1 to 50 of VP 1, or corresponding to aminoacid positions 261, 381, 447, 534, 573, and/or 587 of VP1, preferably toamino acid position 447 or
 587. 109. The library according to claim 94,wherein the structural gene, preferably the cap gene has at least onefurther mutation leading to for example at least one point mutation, atleast one internal deletion, insertion and/or substitution of one orseveral amino acids or at least one N- or C-terminal deletion, insertionand/or substitution of one or several amino acids, or a combination ofthese mutations, preferably a mutation inhibiting heparansulfateproteoglycan binding, integrin and/or Fibroblast Growth Factor Receptor(FGFR) binding.
 110. The library according to claim 94, wherein thestructural gene, preferably the cap gene has a further constantinsertion of at least one codon upstream and/or downstream of theinsertion site of the inserted nucleic acid sequence, preferably of oneor two or three codons coding for Ala, Gly, Leu, Ile, Asp and/or Arg,especially an insertion of three Ala upstream and two Ala downstream ofthe insertion site.
 111. A library of nucleic acids comprising amultiplicity of expressible structural genes from at least oneeukaryotic virus, obtainable by the method of claim
 77. 112. A libraryof virions, especially parvovirus virions, with capsid proteinmodifications.
 113. The library of virions according to claim 112,containing particles containing the genetic information necessary togenerate viral progeny.
 114. The library of virions according to claim113, where each particle contains the genetic information necessary togenerate viral progeny.
 115. The library of virions according to claim112 generated by using a set of nucleic acids, each encoding at leastone structural gene from a eukaryotic virus and comprising a suitablepackaging sequence.
 116. The library of virions according to claim 112,obtainable by expressing the nucleic acids of a library of nucleic acidscomprising a multiplicity of expressible structural genes from at leastone eukaryotic virus.
 117. A nucleic acid encoding a cap gene comprisingat least one recombination site within the cap gene, preferably for theGateway or cre/lox system preferably after amino acid position 587 ofVP1 wherein the inserted nucleic acid sequences are inserted after anucleic acid corresponding to any site within the first amino-terminalamino acids 1 to 50 of VP 1, or corresponding to amino acid positions261, 381, 447, 534, 573, and/or 587 of VP1, preferably to amino acidposition 447 or 587, or comprising at least one endonuclease restrictionsite or polylinker that is not present in the respective wildtype geneand that is inserted after a nucleic acid corresponding to any sitewithin the first amino-terminal amino acids 1 to 50 of VP 1, orcorresponding to amino acid positions 261, 381, 447, 534, 573, and/or587 of VPI, preferably to amino acid position 447 or 587, or encoding acap gene with a sequence of the plasmid pWT99oen.
 118. The nucleic acidencoding a cap gene of claim 117, wherein such recombination site,endonuclease restriction site or polylinker further contains a stopcodon.
 119. The nucleic acid encoding a cap gene according to claim 117,wherein such cap gene has at least one mutation leading to for exampleat least one point mutation, at least one internal deletion, insertionand/or substitution of one or several amino acids or at least one N- orC-terminal deletion, insertion and/or substitution of one or severalamino acids, or a combination of these mutations.
 120. The nucleic acidencoding a cap gene according to claim 117, wherein the cap gene has afurther constant insertion of at least one codon upstream and/ordownstream of the insertion site of the inserted nucleic acid sequence,preferably of one or two or three codons coding for Ala, Gly, Leu, Ile,Asp and/or Arg, especially an insertion of three Ala upstream and twoAla downstream of the insertion site.
 121. (Currently Amended) A nucleicacid encoding a cap gene, wherein such cap gene has an insertion leadingto amino acids comprising an RGD or DDD motif, preferably an RGDXP (SEQID NO: 1) or DDDXP (SEQ ID NO: 2) motif, especially an RGD motif that isnot present in human proteins, excluding the insertion AGTFALRGDNPQG(SEQ ID NO: 3), or leading to amino acids RGDXXXX (SEQ ID NO: 4),RGDXPXX (SEQ ID NO: 5), DDDXPXX (SEQ ID NO: 6), RGDAVGV (SEQ ID NO: 7),RGDTPTS (SEQ ID NO: 8), RSNAVVP (SEQ ID NO: 13), RDNAVVP (SEQ ID NO:10), GKLFVDR (SEQ ID NO: 9), GENQARS (SEQ ID NO: 11), RSNGVVP (SEQ IDNO: 12), or NSVRAPP (SEQ ID NO: 14), or which is the nucleotidicsequence GANGANNACNNNNCNANNANN (N=A,C,G or T; SEQ ID NO: 15) or aninsertion comprising that sequence.
 122. The nucleic acid according toclaim 121, wherein the inserted nucleic acid sequences are inserted atany site corresponding to the first amino-terminal amino acids 1 to 50of VP1, after corresponding amino acid positions 261, 381, 447, 534,573, and/or 587 of VP1, preferably after amino acid position 447 or 587.123. The nucleic acid according to claim 121, wherein such cap gene hasat least one mutation leading to for example at least one pointmutation, at least one internal deletion, insertion and/or substitutionof one or several amino acids or at least one N- or C-terminal deletion,insertion and/or substitution of one or several amino acids, or acombination of these mutations.
 124. The nucleic acid according to claim121, wherein the cap gene has a further constant insertion of at leastone codon upstream and/or downstream of the insertion site of theinserted nucleic acid sequence, preferably of two or three codons codingfor Ala, Gly, Leu, Ile, Asp and/or Arg, preferably an insertion of threeAla upstream and two Ala downstream of the insertion site.
 125. The useof a nucleic acid encoding a cap gene according to claim 117 for thepreparation of a library of nucleic acids comprising a multiplicity ofexpressible cap genes from at least one eukaryotic virus, preferably aparvovirus.
 126. The use of a nucleic acid encoding a cap gene accordingto claim 121 for the preparation of a library of nucleic acidscomprising a multiplicity of expressible cap genes from at least oneeukaryotic virus, preferably a parvovirus.
 127. A vector constructcomprising a nucleic acid according to claim
 117. 128. A vectorconstruct comprising a nucleic acid according to claim
 121. 129. Abacterium or a cell comprising a nucleic acid according to claim 117.130. A bacterium or a cell comprising a nucleic acid according to claim121.
 131. A method for the selection of a recombinant virion with anincreased infectivity or specificity for a specific cell type comprisingthe steps of i) providing at least one first cell with a vectorconstruct comprising at least one nucleic acid from the libraryaccording to claim 94 together with ITRs and an gene providing necessaryfunctions of a Rep protein for the packaging of a virion; ii) providingsuch first cell with necessary cellular, viral, physical and/or chemicalhelper functions for the packaging of a virions; iii) incubating suchfirst cell under suitable conditions for the packaging of virions andcollecting produced virions by such first cell iv) infecting at leastone second cell with such virions v) providing such second cell withnecessary cellular, viral, physical and/or chemical helper functions forthe packaging of a virion; vi) incubating such second cell undersuitable conditions for the packaging of virions and collecting producedvirions by such second cell; whereas steps iv) to vi) can be repeatedseveral times.
 132. The method of claim 131 additionally comprising thesteps vii) infecting at least one third cell with the collected virions,whereas such third cell is not permissive for such virions, and viii)collecting the virions that did not infect such third cell.
 133. Amethod for the selection of a recombinant virion with a modifiedimmunogenicity comprising the steps of i) providing at least one firstcell with a vector construct comprising at least one nucleic acid fromthe library of the invention according to claim 94 together with asecond nucleic acid necessary for the packaging of virion. ii) providingsuch first cell with necessary cellular, viral, physical and/or chemicalhelper functions for the packaging of virions if necessary; iii)incubating such first cell under suitable conditions for the packagingof virions and collecting produced virions by such first cell; iv)applying an immunoselection step to the produced virions; v) infectingat least one first or second cell with such collected virions; vi)providing such first or second cell with necessary cellular, viral,physical and/or chemical helper functions for the packaging of a virion;vii) incubating such first or second cell under suitable conditions forthe packaging of virions and collecting produced virions by such firstor second cell; whereas steps iv) to vii) can be repeated several times.134. The method of claim 133, whereas the immunoselection step is apreincubation of the produced virions with monoclonal or polyclonalantibodies or an immunodepletion reaction.
 135. A method for theidentification of a mutant cap gene comprising the steps of claim 131and in addition the step of ix) cloning the nucleic acid of the capgene(s) of the virion.
 136. A method for the identification of a mutantcap gene comprising the steps of claim 133 and in addition the step ofix) cloning the nucleic acid of the cap gene(s) of the virion.
 137. Themethod according to claim 131, comprising in addition a step forselection of virions, e.g. an affinity binding step of virions, an ionexchange chromatography step or an immuno-selection step.
 138. Themethod according to claim 133, comprising in addition a step forselection of virions, e.g. an affinity binding step of virions, an ionexchange chromatography step or an immuno-selection step.
 139. A methodfor the selection of a receptor binding motif comprising the steps asdefined in claim 131, wherein such second cell expresses the respectivereceptor, preferably recombinantly expresses or over-expresses suchreceptor.
 140. A method for the selection of a receptor binding motifcomprising the steps as defined in claim 133, wherein such second cellexpresses the respective receptor, preferably recombinantly expresses orover-expresses such receptor.
 141. A method for the in vivo selection ofa recombinant virion capable of infecting a specific cell typecomprising the steps of i) providing at least one first cell with avector construct comprising at least one nucleic acid from the libraryaccording to claim 94 together with ITRs and a gene providing necessaryfunctions of a Rep protein for the packaging of a virion; ii) providingsuch first cell with necessary cellular or viral helper functions forthe packaging of a virion; iii) incubating such first cell undersuitable conditions for the packaging of virions and collecting producedvirions by such first cell; iv) infecting an animal with such virions.142. A method for the identification of a mutant cap gene leading tovirions having an increased infectivity or specificity for a specificcell type comprising the steps of claim 141 and in addition the step ofv) cloning the nucleic acid of the cap gene(s) from such cell type ofthe animal.
 143. A Cap protein, encoded by the nucleic acids accordingto claim
 121. 144. A polypeptide comprising a peptide with the sequenceRGDAVGV, (SEQ ID NO: 7) RGDTPTS, (SEQ ID NO: 8) GKLFVDR, (SEQ ID NO: 9)RDNAVVP, (SEQ ID NO: 10) GENQARS, (SEQ ID NO: 11) RSNGVVP, (SEQ ID NO:12) RSNAVVP (SEQ ID NO: 13) or NSVRAPP. (SEQ ID NO: 14)


145. The polypeptide according to claim 144, consisting of a peptidewith the sequence RGDAVGV (SEQ ID NO: 7), RGDTPTS (SEQ ID NO: 8),GKLFVDR (SEQ ID NO: 9), RDNAVVP (SEQ ID NO: 10), GENQARS (SEQ ID NO:11), RSNGVVP (SEQ ID NO: 12), RSNAVVP (SEQ ID NO: 13) or NSVRAPP (SEQ IDNO: 14).
 146. The polypeptide according to claim 144, wherein thepolypeptide is a Cap polypeptide, preferably derived from a parvovirus,especially from an AAV.
 147. A method for the retargeting of eukaryoticviruses, preferably parvoviruses, especially AAV, the method comprisingusing a polypeptide according to claim 144, comprising or consisting ofa peptide with the sequence RGDXXXX (SEQ ID NO: 4), RGDXPXX (SEQ ID NO:5), or DDDXPXX (SEQ ID NO: 6), with the exception of AGTFALRGDNPQG (SEQID NO: 3).
 148. A recombinant virion obtainable by the method accordingto claim
 131. 149. A recombinant virion obtainable by the methodaccording to claim
 133. 150. A mutant cap gene obtainable by the methodaccording to claim
 135. 151. A mutant cap gene obtainable by the methodaccording to claim
 136. 152. A mutant cap gene obtainable by the methodaccording to claim
 151. 153. A Cap protein encoded by a mutant cap geneaccording to claim
 150. 154. A Cap protein encoded by a mutant cap geneaccording to claim
 151. 155. A Cap protein encoded by a mutant cap geneaccording to claim
 152. 156. A virion comprising a Cap protein accordingto claim
 153. 157. A virion comprising a Cap protein according to claim154.
 158. A virion comprising a Cap protein according to claim
 155. 159.A medicament for the treatment of a patient suffering from cancer, anautoimmune disease, an infectious disease, or a genetic defectcomprising a construct selected from the group consisting of a virion asdefined in claim 148, a virion as defined in claim 149, a virion asdefined in claim 156, a virion as defined in claim 157, a virion asdefined in claim 158, a cap gene as defined in claim 150, a cap gene asdefined in claim 151, a cap gene as defined in claim 152, a Cap proteinas defined in claim 153, a Cap protein as defined in claim 154, or a Capprotein as defined in claim
 155. 160. A method for treating a patientsuffering from cancer, an autoimmune disease, an infectious disease, ora genetic defect comprising administering to the patient a constructselected from the group consisting of a virion as defined in claim 148,a virion as defined in claim 149, a virion as defined in claim 156, avirion as defined in claim 157, a virion as defined in claim 158, a capgene as defined in claim 150, a cap gene as defined in claim 151, a capgene as defined in claim 152, a Cap protein as defined in claim 153, aCap protein as defined in claim 154, or a Cap protein as defined inclaim 155.